Tissue resident memory CD4⁺T cells are sustained by site-specific levels of self-renewal and continuous replacement

Tissue-resident memory T cells (T_RM) protect from repeat infections within organs and barrier sites. The breadth and duration of such protection is defined at minimum by three quantities; the rate at which new T_RMare generated from precursors, their rate of self-renewal, and their rate of loss through death, egress, or differentiation. Quanti-fying these processes individually is challenging. Here we combine genetic fate mapping tools and mathematical models to untangle these basic homeostatic properties of CD4⁺T_RMin the skin and gut lamina propria (LP) of healthy adult mice. We show that CD69⁺CD4⁺T_RMin skin reside for ∼24 days and self-renew more slowly, such that clones halve in size approximately every 5 weeks; and approximately 2% of cells are replaced daily from precursors. CD69⁺CD4⁺T_RMin LP have shorter residencies (∼14 days) and are maintained largely by immigration (4-6% per day). We also find evidence that the continuous replacement of CD69⁺CD4⁺T_RMat both sites derives from circulating effector-memory CD4⁺T cells, in skin possibly via a local CD69⁻intermediate. Our approach maps the ontogeny of CD4⁺T_RMin skin and LP and exposes their dynamic and distinct behaviours, with continuous seeding and erosion potentially impacting the duration of immunity at these sites.